Measuring RNA transcript levels

Research problem

– A small RNA, RteR, inhibits transfer of the 65 kb conjugative transposon CTnDOT

attR attL

ermF tetQexcision regulatory transfer

oriTintDOT

mob

PcPE

RteR

PT

Question: precisely where in the transfer region is RteR binding???

Possible means of measuring transcript levels

• Promoter fusions

• Qualitative RT-PCR

• Quantitative or “real time”: RT-qPCR

• Northern blotting analysis

• 5’ and 3’ RACE – lengths of transcript rather than abundance

Promoter fusions

• A reporter gene (e.g. lacZ) is fused to a promoter of interest to test for transcriptional activity

• This represents only activity of promoter, and thus excludes other levels of transcriptional regulation

lacZ-35 -10

Reverse transcriptase (RT)-PCR

• A.k.a. qualitative or endpoint

• RT is “reverse transcriptase”: synthesize cDNA from total RNA pools– Generally isolate RNA from different cell

treatments, e.g.:• Carbohydrate source• Aerobic vs. anaerobic growth• Growth phase• With and/or without gene of interest in trans

Transcript levels +/- small RNA, RteR

Ptra

A B C D E F G H I J K L M N O P Q

traA traB-C traD traE-F traF-G

- + - + - + - + - +RteR

Quantitative or “real time” PCR

• Gives you information in real time after each cycle

• For obvious reasons a quantitative value, rather than a subjective relative value, can be more interesting and meaningful

A typical plot from a qPCR run

Melt curves to ensure primer quality

Calculating when a sample crosses a given threshold (Ct or Cq) to quantify transcript abundance

Caveats to qPCR

• Experimental methods/calculations can greatly skew results, as with any technique

• Many have false sense of trust in the word “quantitative” believe technique is inherently more accurate

Northern blotting

• Another means of (literally) visualizing transcript levels

• Can detect RNA processing events that would other wise be unnoticed in RT-PCR

• Caveat is that it is qualitative, and there is a limit to transcript size due to electrophoresis capabilities

RteR Northern blotting analysis

Marker

BT4001 ΩQABpJW305- Tc

EmptyVector

150

100

90

8070

60

50

40

30

T7 promoter

T7 promoter

PCR

In vitro transcriptionreaction

+

32P-αUTP

Antisense ssRNA probe to region of interest

DecadeMarker

BT4001 ΩQAB pJW305

- Tc + Tc

150

100

80

60

40

Can detect multiple transcripts

RACE analysis to identify 5’ and 3’ ends of RteR

Total RNA5’ RACE

5’-P

+ TAP - TAP

5’-PPP

5’-PPP5’-P5’-P 5’-P

cDNA synthesisPCR

+ TAP - TAP

5’-PPP

adapter ligation

3’ RACE

OH-3’

calf. Int. phos.adapter ligation

cDNA synthesisPCR

processed end+ TAP - TAP

primary end